Basement membranes are extracellular membranes structures consisting of collagen IV, laminin, nidogen and heparan sulfate proteoglycan as major components and are deposited by epithelium (or endothelium) at interfaces to the interstitium. The basement membranes play essential roles in normal embryogenesis, tissue remodeling (would healing), and filtration process. They are also involved in such processes as scar formation, thickening and dysfunction of capillary and glomerular basement membrane with age and diabetic condition, and fibrosis in liver and cornea. Al of these abnormalities can be attributed to the malformation of the basement membrane. The long range goal of this proposal is to elucidate the mechanism for the formation and maintenance of basement membrane in vivo and in vitro, with a special focus on the tissue specific transcriptional regulation of collagen IV genes. Progress has been made recently toward this objective with the identification of several transcriptional enhancers of the mouse collagen IV genes in the COL4a1 first intron and in the COL4a2 third intron. One of the enhancers (0.3L), in the first intron of COL4a1 gene, has been characterized and found to function only in collagen IV producing cells. It has also been found to be regulated in a differentiation induced manner in the F9 cell system which models the early mouse development. Furthermore, two GAACAAT elements of the 0.3L enhancer, which appear to be new cis-acting elements, are essential for the tissue specific activity of the 0.3L enhancer. On the other hand the COL4a2 third intron enhancers have not been characterized in depth. This proposal is specially intended to obtain support for the completion of the characterization of the COL4a2 third intron enhancers. Toward this objective the proposed specific aims are: 1) To characterize the cis-acting elements responsible for the transcriptional enhancing activity of the COL4a2 third intron, and 2) To characterize, purify and clone the trans-acting factors that bind to the cis-acting elements of the mouse COL4a2 third intron enhancer. These aims will be pursued using molecular biological technique such as deletion analysis, mutational analysis, transfection, and molecular cloning as well as biochemical approach for protein purification.